Fluorescent tRNA Derivatives and Ribosome Recognition

Abstract

The interactions of tRNA’s with aminoacyl-tRNA synthetases and ribosomes have been studied rather extensively using a variety of approaches (Rich and RajBhandary 1976; Altaian 1978; Schimmel et al. 1979). For a number of years we have investigated the chemistry of introducing fluorescent groups into tRNA in order to use the fluorescent tRNA’s as tools in the study of aminoacyl-tRNA synthetase and ribosome recognition (Wintermeyer and Zachau 1971, 1979). The procedure eventually applied allows the specific replacement of odd bases in internal positions of the tRNA molecule with fluorescent dyes. The replacement basically involves a two-step reaction: a ribosylic aldehyde group is created in the tRNA by selective excision of a base which subsequently can be condensed with a fluorophor possessing either a primary amino or a hydrazino group (Fig. 1). For the experiments described below fluorescent derivatives of tRNA^Phe (yeast) have been used in which wybutine (position 37, next to the anticodon) or dihydrouracil (positions 16 or 17 in the D-loop) had been replaced with ethidium (Etd) or proflavin (Prf). Upon modification the tRNA^Phe retained its activity in the partial reactions of protein synthesis, indicating that the essential elements of the native structure are not disturbed by insertion of the dyes. In support of this conclusion, the direct structural investigation of tRNA ^Phe_Prf by NMR spectroscopy has shown that the insertion of Prf reverses the substantial changes of the spectrum which are introduced by the excision of wybutine from tRNA^Phe (Wong et al. 1975).